Proposals set forth in this renewal grant application represent a continuation of our ongoing delineation of the role papillomaviruses (PV) play in the genesis of genital benign tumors and their subsequent progression to malignancies. While previous studies have attempted to relate the biological properties of benign (HPV 6 and HPV 11) and oncogenic (HPV 16 and HPV 18) papillomaviruses, these studies have always suffered from the fact that the analyses involved gene or subgenic regions taken out of the context of the entire genome. The vast differences between the nucleotide sequences of these two groups of papillomaviruses throughout their genomes precludes this contextual analysis. We have identified two genetic variants of HPV 11 which exhibit vastly different oncogenic potential both in vivo and in vitro. Our nucleotide sequence analysis of the early regions of these two variants identified 16 nucleotide differences including silent amino acid changes, altered amino acids, and substitutions in the upstream regulatory region (URR) which control viral transcription. The major objective of this renewal grant application is to delineate the molecular basis for the differential oncogenicity of benign and malignant variants of HPV 11. As none of the sequence variations observed occur in either the E6 and E7 open reading frames thought to be involved in papillomavirus oncogenesis, the molecular basis for the observed differential oncogenic properties of these variants must lie elsewhere within the HPV 11 genome. Several possibilities exists that either individually or in concert may affect the differential oncogenic properties of these HPV 11 mutants and include: 1) alteration of early gene expression due to variations in the URR which may respond to cellular factors or variations in the E2 open reading frame (ORF); 2) the enhancement of another putative oncogene such as E5; 3) or an alteration of a gene (E1) which has been shown to be important for viral plasmid maintenance and replication. In this proposal the functional differences of the oncogenic and non-oncogenic variants of HPV 11 will be molecularly defined by a combination of site-directed mutagenesis and exchanges of specific genetic sequences between the HPV 11 variants. These studies will also include an the analysis of the effects of the in vitro transforming capabilities of the E5 ORF of HPV 11P and HPV 11VC and the effect of this gene on two important induction pathways. In addition we will also include an examination of the regulation of viral expression, and the relative transforming functions of oncogenic and nononcogenic HPV 11 variants and should shed light on the nature of the genetic influences that are involved in malignant progression. The methodologies to achieve these objectives are all currently in use in our laboratory including a variety of physicochemical, enzymological and hybridization techniques for DNA, RNA, and protein and expression analysis.